Computational modeling of differences in the quorum sensing induced luminescence phenotypes of Vibrio harveyi and Vibrio cholerae

Vibrio harveyi and Vibrio cholerae have quorum sensing pathways with similar design and highly homologous components including multiple small RNAs (sRNAs). However, the associated luminescence phenotypes of strains with sRNA deletions differ dramatically: in V. harveyi, the sRNAs act additively; however, in V. cholerae, the sRNAs act redundantly. Furthermore, there are striking differences in the luminescence phenotypes for different pathway mutants in V. harveyi and V. cholerae. However, these differences have not been connected with the observed differences for the sRNA deletion strains in these bacteria. In this work, we present a model for quorum sensing induced luminescence phenotypes focusing on the interactions of multiple sRNAs with target mRNA. Within our model, we find that one key parameter - the fold-change in protein concentration necessary for luminescence activation - can control whether the sRNAs appear to act additively or redundantly. For specific parameter choices, we find that differences in this key parameter can also explain hitherto unconnected luminescence phenotypes differences for various pathway mutants in V. harveyi and V. cholerae. The model can thus provide a unifying explanation for observed differences in luminescence phenotypes and can also be used to make testable predictions for future experiments.     

Isolation and identification of quorum quenching bacteria from environmental samples

A large number of Gram-negative pathogens produce N-acylhomoserine lactones (AHLs) as signal molecules for quorum sensing (QS). This cell-cell communication system allows them to coordinate gene expression and regulate virulence. Therefore, strategies to inhibit QS are promising for the control of infectious diseases. The aim of the present study was to develop a high-throughput method for the isolation and identification of AHL-degrading bacteria from environmental samples. Samples were cultured in a microtitre plate in a minimal medium containing 1 mM N-(3-oxo-dodecanoyl)-l-homoserine lactone and 2 mM N-(3-oxo-hexanoyl)-l-homoserine lactone as the sole sources of carbon and nitrogen. Isolates growing on this minimal medium were subcultured and identified by partial 16S rRNA gene sequencing. Subsequently, the AHL-degrading capacity of each isolate was evaluated in the Pseudomonas aeruginosa QSIS2 biosensor assay, as such or after treatment with heat or proteinase K. The 16 samples tested yielded a total of 59 isolates which are, either alone or as part of a consortium, able to use AHL signal molecules as sole sources of carbon and nitrogen. Follow-up experiments have shown that in each sample there is at least one isolate with quorum quenching (QQ) activity, and that for all samples combined, 41 isolates have QQ activity. Furthermore, heat treatment did not fully inhibit QQ activity in all isolates. In some isolates, QQ activity was lost after proteinase K treatment, while others remained able to quench QS. Therefore, it is likely that some isolates produce and secrete (a) heat-stable, low molecular weight inhibitory compound(s).     

Synthesis and application of an N-acylated l-homoserine lactone derivatized affinity matrix for the isolation of quorum sensing signal receptors

The design and synthesis of an agarose resin functionalized with a Gram-negative quorum sensing (QS) signaling molecule analogue is described. The modified resin was utilized in affinity pull-down assays to successfully isolate QscR, a LuxR-type QS receptor from Pseudomonas aeruginosa. This resin may facilitate the identification of novel QS signal receptors using affinity chromatography techniques.     

Response surface optimized peroxyoxalate chemiluminescence of octahydro‐Schiff base derivative as new luminophor and study of the quenching effect of some cations,amino acids and cholesterol

We report the first detailed study of the characteristics of an octahydro‐Schiff base derivative as a new luminophor in the peroxyoxalate chemiluminescence (POCL) system. The effect of reagents on this new POCL system was investigated. In addition, the response surface methodology was used for evaluating the relative significance of variables in this POCL system, establishing models and determining optimal conditions. The quenching effect of some cations and compounds such as Cu²⁺, Fe³⁺, Hg²⁺, imidazole, histidine and cholesterol on an optimized POCL reaction were studied. The dynamic ranges were up to approximaterly 100 and 175 × 10^?6 M for Cu²⁺ and cholesterol respectively. The detection limits were 3.3 × 10^?6 m and 2.58 × 10^?6 m for Cu²⁺ and histidine, respectively. In all cases the relative standard deviations were 4–5% (n = 4). Copyright © 2014 John Wiley & Sons, Ltd.     

Single cell time-resolved quorum responses reveal dependence on cell density and configuration

Bacterial communication via quorum sensing has been extensively investigated in recent years. Bacteria communicate in a complex manner through the production, release, and reception of diffusible low molecular weight chemical signaling molecules. Much work has focused on understanding the basic mechanisms of quorum sensing. As more and more bacteria grow resistant to conventional antibiotics, the development of drugs that do not kill bacteria but instead interrupt their communication is of increasing interest. This study presents a method for analyzing bacterial communication by investigating single cell responses. Most conventional analysis methods for bacterial communication are based on the averaged response from many bacteria, masking how individual cells respond to their immediate environment. We applied a fiber-optic microarray to record cellular communication from single cells. Single cell quorum sensing systems have previously been employed, but the highly ordered array reported here is an improvement because it allows us to simultaneously investigate cellular communication in many different environments with known cellular densities and configurations. We employed this method to detect how genes under quorum regulation are induced or repressed over time on the single cell level and to determine whether cellular density and configuration are indicative of the single cell temporal patterns of gene expression.     

Evidence on how a conserved glycine in the hinge region of HapR regulates its DNA binding ability: lessons from a natural variant

HapR has been recognized as a quorum-sensing master regulator in Vibrio cholerae. Because it controls a plethora of disparate cellular events, the absence of a functional HapR affects the physiology of V. cholerae to a great extent. In the current study, we pursued an understanding of an observation of a natural protease-deficient non-O1, non-O139 variant V. cholerae strain V2. Intriguingly, a nonfunctional HapR (henceforth designated as HapR(V2)) harboring a substitution of glycine to aspartate at position 39 of the N-terminal hinge region has been identified. An in vitro gel shift assay clearly suggested the inability of HapR(V2) to interact with various cognate promoters. Reinstatement of glycine at position 39 restores DNA binding ability of HapR(V2) (HapR(V2G)), thereby rescuing the protease-negative phenotype of this strain. The elution profile of HapR(V2) and HapR(V2G) proteins in size-exclusion chromatography and their circular dichroism spectra did not reflect any significant differences to explain the functional discrepancies between the two proteins. To gain insight into the structure-function relationship of these two proteins, we acquired small/wide angle x-ray scattering data from samples of the native and G39D mutant. Although Guinier analysis and indirect Fourier transformation of scattering indicated only a slight difference in the shape parameters, structure reconstruction using dummy amino acids concluded that although HapR adopts a "Y" shape similar to its crystal structure, the G39D mutation in hinge drastically altered the DNA binding domains by bringing them in close proximity. This altered spatial orientation of the helix-turn-helix domains in this natural variant provides the first structural evidence on the functional role of the hinge region in quorum sensing-related DNA-binding regulatory proteins of Vibrio spp.     

Processing the interspecies quorum-sensing signal autoinducer-2 (AI-2): characterization of phospho-(S)-4,5-dihydroxy-2,3-pentanedione isomerization by LsrG protein

The molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD) is produced by many different species of bacteria and is the precursor of the signal molecule autoinducer-2 (AI-2). AI-2 mediates interspecies communication and facilitates regulation of bacterial behaviors such as biofilm formation and virulence. A variety of bacterial species have the ability to sequester and process the AI-2 present in their environment, thereby interfering with the cell-cell communication of other bacteria. This process involves the AI-2-regulated lsr operon, comprised of the Lsr transport system that facilitates uptake of the signal, a kinase that phosphorylates the signal to phospho-DPD (P-DPD), and enzymes (like LsrG) that are responsible for processing the phosphorylated signal. Because P-DPD is the intracellular inducer of the lsr operon, enzymes involved in P-DPD processing impact the levels of Lsr expression. Here we show that LsrG catalyzes isomerization of P-DPD into 3,4,4-trihydroxy-2-pentanone-5-phosphate. We present the crystal structure of LsrG, identify potential catalytic residues, and determine which of these residues affects P-DPD processing in vivo and in vitro. We also show that an lsrG deletion mutant accumulates at least 10 times more P-DPD than wild type cells. Consistent with this result, we find that the lsrG mutant has increased expression of the lsr operon and an altered profile of AI-2 accumulation and removal. Understanding of the biochemical mechanisms employed by bacteria to quench signaling of other species can be of great utility in the development of therapies to control bacterial behavior.     

Effect of transition metal ions on the fluorescence and Taq-catalyzed polymerase chain reaction of tricyclic cytidine analogs

The cytosine analogs 1,3-diaza-2-oxophenothiazine (tC) and 1,3-diaza-2-oxophenoxazine (tCo) stand out among fluorescent bases due to their unquenched fluorescence emission in double-stranded DNA. Recently, we reported a method for the generation of densely tCo-labeled DNA by polymerase chain reaction (PCR) that relied on the use of the extremely thermostable Deep Vent polymerase. We have now developed a protocol that employs the more commonly used Taq polymerase. Supplementing the PCR with Mn²⁺ or Co²⁺ ions dramatically increased the amount of tCo triphosphate (dtCoTP) incorporated and, thus, enhanced the brightness of the PCR products. The resulting PCR products could be easily detected in gels based on their intrinsic fluorescence. The Mn²⁺ ions modulate the PCR by improving the bypass of template tCo and the overall catalytic efficiency. In contrast to the lower fidelity during tCo bypass, Mn²⁺ improved the ability of Taq polymerase to distinguish between dtCoTP and dTTP when copying a template dA. Interestingly, Mn²⁺ ions hardly affect the fluorescence emission of tC(o), whereas the coordination of Co²⁺ ions with the phosphate groups of DNA and nucleotides statically quenches tC(o) fluorescence with small reciprocal Stern–Vollmer constants of 10–300 μM.     

信号分子AI-2的检测方法研究进展

很多细菌在生长过程中会产生一些小分子量的自诱导分子,也称为信号分子,当其随着细胞数量增加而积累到一定阈值时能够调控细菌特定基因的表达,这个过程称为群体感应(Quorum sensing,QS)。多数自诱导分子具有物种特异性,但很多种属的细菌都会产生一种共同的自诱导分子AI-2,AI-2被认为是细菌种间交流的通用语言。定量检测AI-2对于研究与其相关的生理生化过程是非常必要的。然而,目前还没有一种标准的定量检测AI-2的方法。因此,本文就目前关于AI-2的检测方法进行综述,为后续研究者提供参考。     

氧脂素对赭曲霉在大豆培养基质中合成赭曲霉毒素A的影响

【背景】赭曲霉毒素A (ochratoxin A,OTA)是曲霉属和青霉属等真菌的次级代谢产物,严重威胁农产品及食品安全,氧脂素羟基十八碳二烯酸(hydroxyoctadecaenoic acids,HODEs)被认为可能是曲霉属的群体感应信号分子,调节曲霉的生长发育和次级代谢物生成。【目的】主要研究HODEs对赭曲霉(Aspergillusochraceus)菌株AS3.4412产生OTA的影响,检测孢子密度、培养基类别以及内、外源HODEs作用下OTA产量的不同变化。【方法】分别在PDB、黄豆和黑豆培养基中进行赭曲霉的培养,采用高效液相色谱-荧光检测法测定OTA含量,采用高效液相色谱-质谱法测定氧脂素含量,根据变化规律寻找赭曲霉群体密度、氧脂素、OTA三者间的关系。【结果】低密度赭曲霉培养物(103 spores/mL)中9(S)-HODE/13(S)-HODE及OTA产量高于高密度赭曲霉(106 spores/mL);外源添加9(S)-HODE能促进OTA合成,13(S)-HODE可以抑制OTA合成;赭曲霉侵染抗氧化能力更高的黑豆产生更多的OTA。【结论】OTA的合成受到赭曲霉群体密度和氧脂素的影响,推测9(S)-HODE和13(S)-HODE是赭曲霉群体感应信号分子,并且二者在调节OTA合成中具有相反的作用。